Labeled circular DNA molecules for analysis of DNA topology, and topoisomerases and for drug screening

ABSTRACT

The present invention provides labeled circular plasmid DNA molecules for studying DNA topology and topoisomerases. The molecules of the present invention also provide tools for high throughput drug screening for inhibitors of DNA gyrases and DNA topoisomerases for anticancer drug discovery and antibiotics discovery.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation application of U.S. Ser. No.15/286,046, filed Oct. 5, 2016, which claims the priority benefit ofU.S. provisional application Ser. No. 62/237,236, filed Oct. 5, 2015,both of which are incorporated herein by reference in their entirety.

The Sequence Listing for this application is labeled“SeqList-29Sep16.txt”, which was created on Sep. 29, 2016, and is 4 KB.The entire content is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

DNA topology plays essential roles in several fundamental biologicalprocesses, such as DNA replication, recombination, and transcription.DNA topology is a tightly-regulated property of the DNA double helixthat affects genomic stability and influences susceptibility to cancerand certain hereditary diseases, such as fragile X syndrome and autism.DNA topoisomerases that control DNA topology inside cells are importanttargets of anti-cancer drugs, such as camptothecin and doxorubicin, andantibacterial agents, such as ciprofloxacin.

Typically agarose gel electrophoresis is employed to study DNA topology.Because gel electrophoresis is time-consuming and labor intensive, it isdesirable to develop other methods, such as fluorescence-based methods,for such studies. For example, fluorescence dyes, such as PicoGreen (1),have been shown to differentially bind to supercoiled (sc) and relaxed(rx) DNA molecules to yield different fluorescence properties. Thesefluorescence dyes were used to study DNA topoisomerases; however, thedifference of the fluorescence intensity of the dyes binding to sc andrx DNA is too small to be widely used to study the properties of DNAtopoisomerases and to screen inhibitors against these topoisomerases(1).

Another type of assay was developed based on a unique property of sc DNAmolecules that prefer binding to triplex-form oligomers if the scplasmids contain one or multiple triplex-forming sequences (2, 3).Maxwell and coworkers utilized a method in which an immobilizedtriplex-forming oligomer more efficiently captured sc plasmids than rxplasmids (2). The captured plasmids could subsequently be quantified bya DNA-binding dye, such as SYBR Green. However, this method requiresimmobilization of oligomer to a solid surface, filtration, and multiplewashing steps. Because streptavidin-coated 1526-well plates are notcommercially available, this method is not compatible with ultra-highthroughput screening to identify gyrase inhibitors from small compoundlibraries using 1526-well plates.

Another method, also based on the triplex-forming oligomers, wasdeveloped by using fluorescence anisotropy for the readout (3).Nevertheless, the signal to noise ratio is a concern and an expensivefluorimeter with the capacity to measure fluorescence anisotropy isrequired (3).

More recently, Berger and coworkers made a circular plasmid DNA templatethat contains a fluorophore (fluorescein) and quencher (dabcyl) onopposite strands of a double-stranded DNA molecule and developed areal-time assay to study DNA topological changes with this fluorescentlylabeled DNA (4). However, the production involving two steps offluorophore and quencher insertion into the DNA result in a low yield offluorescently labeled DNA, which low yield is cost prohibitive andimpedes wide use of the assay to study DNA topology, topoisomerases, andto screen compounds against DNA topoisomerases (4).

BRIEF SUMMARY OF THE INVENTION

Provided herein are new reagents and methods to mass-producefluorescently labeled circular DNA molecules with high yields to studyDNA topology and topoisomerases by fluorescence resonance energytransfer (FRET), and to screen anti-cancer drugs and antibioticstargeting DNA topoisomerases using high throughput drug screening.

Reagents provided include novel nucleic acid sequences comprising anadenosine-thymidine dinucleotide repeat (AT)_(n) sequence comprising atleast one fluorophore and at least one quencher, each conjugated to adeoxythymidine (dT), wherein the fluorophore-conjugated dT and thequencher-conjugated dT are separated by at least 14 nucleotides. Thenovel nucleic acid sequences provide superior detection efficiency andfacilitate use in high throughput screening.

The subject invention further provides methods for mass-production offluorescently labeled circular DNA molecules with high yields sufficientto study DNA topology and topoisomerases by fluorescence resonanceenergy transfer (FRET) and in high throughput screening to identifynovel anti-cancer drugs and antibiotics targeting DNA topoisomerases.

Further provided herein are methods for screening for inhibitorstargeting DNA topoisomerases, DNA gyrases, DNA nicking endonuclease, DNAendonucleases, and RNAses using the circular plasmid DNA molecules andkits for screening for said inhibitors.

The methods, molecules and kits herein described can be used inconnection with pharmaceutical, medical, and veterinary applications, aswell as fundamental scientific research and methodologies, as would beidentifiable by a skilled person upon reading of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

For a fuller understanding of the nature of the present invention,reference should be had to the following detailed description taken inconnection with the accompanying figures.

FIG. 1A shows a (AT)_(n) DNA of FL905 carrying fluorescein (Fl) anddabcyl (Dab) labels and being able to convert from a hairpin structureto an open structure. The sequence shown represents positions 22 to 63of SEQ ID NO:4.

FIG. 1B shows a model of the FL905 DNA with the Fl and Dab labels inproximity to each other when the (AT)_(n) of FL905 adopts the hairpinstructure.

FIG. 1C shows a model of the FL905 DNA with the Fl and Dab labels beingaway from each other when the (AT)_(n) sequence is in the doublestranded state.

FIG. 2 shows one embodiment of the (AT)_(n) sequence with theDab-labeled deoxythymidine (dT) at the 8th position from the 5′-terminusof the (AT)_(n) and the Fl-labeled dT at the 34th position from the5′-terminus of the (AT)_(n) sequence.

FIG. 3A shows a polyacrylamide gel of FL905 without staining.

FIG. 3B shows fluorescence DNA melting of FL905.

FIG. 3C shows the double-stranded oligomer FL_AT42 carrying a 42 bp ATsequence and two Nt.BbvCI recognition sites (indicated by the arrows)inserted between SphI and BamHI sites of pUC18.

FIG. 3D shows the cloning steps to insert FL_AT42 into SpHI and BamHIsites to yield pAB1 that contains two Nt.BbvCI sites and the 42 bp ATsequence located between the two Nt.BbvCI sites.

FIG. 4A shows the strategy employed to insert FL905 DNA into the (AT)₄₂nucleic acid sequence of pAB1.

FIG. 4B shows the generation of supercoiled (sc) pAB1_FL905 from relaxed(rx) pAB1_FL905 using E. coli DNA gyrase and the relaxation of scpAB1_FL905 using DNA topoisomerase 1.

FIGS. 5A-5B show strategies to study DNA gyrase and to screen inhibitorstargeting DNA gyrases. The inhibition IC₅₀ may be determined by atitration experiment.

FIG. 6 shows strategies to study and screen inhibitors targeting DNAtopoisomerases that relax supercoiled DNA molecules. The Inhibition IC₅₀may be determined by a titration experiment.

FIG. 7 shows strategies to study and screen inhibitors targeting DNAnicking enzymes.

FIG. 8 shows a strategy to construct fluorescently labeled circularplasmid DNA molecules that contain a RNA oligomer inserted between twoNt.BspQI sites.

FIG. 9 shows supercoiled fluorescence-labeled DNA molecules with an RNAoligomer that can be used to study RNase H or other RNases and to screeninhibitors targeting these RNases, whereby supercoiling of the DNAmolecule containing a RNA oligomer is induced with DNA gyrase,relaxation of the supercoiled DNA is induced with RNAse H, andrelaxation is inhibited in the presence of RNAseH inhibitors.

FIG. 10 shows strategies to study and screen inhibitors targetingdouble-stranded DNA endonucleases.

FIG. 11 shows a strategy to construct fluorescently labeled circular DNAmolecules with the fluorescein (FL)-labeled deoxythymidine (dT) on onestrand and the Dabcyl (Dab)-labeled dT on the opposite strand.

FIG. 12 illustrates that the DNA sequences inserted between the Nt.BbvCIand Nb.BbvCI sites on opposite strands contain supercoiling-sensitivestructures such as hairpins and formation of the hairpins leads to highfluorescence because of the distance between the fluorophore and thequencher on the hairpins of opposite DNA strands.

FIG. 13 shows the strategy to produce relaxed (rx) pAB1_FL905.

FIG. 14A shows an agarose gel of pAB1 before (lane 2) and after (lane 3)digestion with Nt.BbvCI.

FIG. 14B shows the annealed FL_905 before (lane 3) and after (lane 2)the ligation reaction with T4 DNA ligase.

FIG. 14C shows the DNA supercoiling assay to convert relaxed (rx)pAB1_FL905 (lane 1) into supercoiled (sc) pAB_FL905 (lane 2). Lane 3 isundigested pAB1_FL905 and lane 4 lambda DNA Hind III digest.

FIGS. 14D-14E show the DNA samples after the relaxed pAB-FL905 waspurified before (14D) and after (14E) ethidium bromide staining. Lane 2is sc pAB1_FL905, lanes 3 and 5 are rx pAB1 FL905 and lanes 4 and 6 arenicked pAB1_FL905.

FIG. 15A shows the fluorescence spectra of sc (red line), rx (blackline), and nicked (blue line) pAB1_FL905.

FIG. 15B shows the kinetics of the nicking reaction by Nt.BbvCI.

FIG. 15C shows the kinetics of the relaxation reaction by E. coli DNAtopoisomerase I.

FIG. 15D shows the kinetics of the supercoiling reaction by E. coli DNAgyrase.

FIG. 16A shows the fluorescence intensity of rx and sc pAB1_FL905,pAB1_FL919, and pAB1_FL920.

FIG. 16B shows fluorescence spectra of sc (red lines) and rx (blacklines) pAB1_FL905.

FIG. 16C shows fluorescence spectra of sc (red lines) and rx (blacklines) pAB1_FL919.

FIG. 16D shows fluorescence spectra of sc (red lines) and rx (blacklines) pAB1_FL920.

FIG. 17A-17B show the sequence of oligomer FL924 that representspositions 22 to 63 of SEQ ID NO:8. FIG. 17A shows the fluorescenceintensity of rx and sc pAB1_FL924 that contains oligomer FL924. FIG. 17Bshows the fluorescence spectra of sc (red lines) and rx (black lines)pAB1_FL924.

FIG. 18A shows DNA supercoiling reactions of pAB1_FL905 in the presenceof novobiocin.

FIG. 18B shows DNA supercoiling reactions of pAB1_FL905 in the presenceof ciprofloxacin.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 shows a first strand of the double-stranded oligomerFL-AT42.

SEQ ID NO:2 shows the second strand of the double-stranded oligomerFL-AT42 including the 5′ and 3′ ends cut with SphI and BamHI.

SEQ ID NO:3 shows the synthetic oligonucleotide FL882.

SEQ ID NO:4 shows the synthetic oligonucleotide FL883.

SEQ ID NO:5 shows the synthetic oligonucleotide FL905.

SEQ ID NO:6 shows the synthetic oligonucleotide FL919.

SEQ ID NO:7 shows the synthetic oligonucleotide FL920.

SEQ ID NO:8 shows the synthetic oligonucleotide FL924.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides fluorophore-quencher nucleic acidmolecules comprising relaxed or supercoiled DNA molecules, theirproduction, optionally, in milligram amounts, and their use to study DNAtopology, DNA topoisomerases, DNA gyrases, DNA nicking endonucleases,DNA endonucleases and RNAses.

Because only a few nanograms of any of the fluorophore-quencher nucleicacid comprising circular DNA plasmids are needed for 384-well or1536-well plates for detection, these DNA plasmids can be used in rapidand efficient high-throughput screening assays to identify inhibitorsfrom the millions of compounds found in small molecule libraries thatpotentially target DNA topoisomerases, DNA gyrases, DNA nickingendonucleases, DNA endonucleases and RNAses.

In accordance with the subject invention, nucleic acids comprising anadenosine-thymidine repeat (AT)_(n) sequence comprising at least onefluorophore and at least one quencher conjugated to a deoxythymidine(dT) of the same strand when present in a circular double-stranded DNAmolecule can be used for fast detection of changes in DNA topology. Ithas also been found that a fluorophore and a quencher conjugated to dTsat a specific distance on the same DNA strand of a double-stranded(AT)_(n) sequence quickly interconvert between an extruded and anunextruded conformation upon supercoiling of the circular DNA. In thesupercoiled state, the (AT)_(n) sequence adopts, for example, a hairpinstructure that brings the fluorophore and the quencher into closeproximity and leads to the quenching of fluorophore fluorescence. In therelaxed circular DNA molecule, where the (AT)_(n) is in adouble-stranded conformation, the fluorophore and quencher are locatedat a sufficient distance such that no quenching occurs and thefluorophore fluoresces.

The instant fluorophore-quencher nucleic acid sequences can comprisealternating adenosine-thymidine (AT)_(n) repeats, or alternativelyG-quadruplexes.

In accordance with the subject invention, large amounts of circularplasmids containing the at least one fluorophore and at least onequencher-comprising nucleic acid can be produced, which large amountsmake the use of the instant nucleic acids in high throughput screeningmethods feasible.

The instant fluorophore-quencher comprising (AT)_(n) nucleic acidsequences have advantageous properties. For example, interconversionbetween the extruded and unextruded conformation of thefluorophore-quencher nucleic acid sequences occurs with fast kineticsallowing rapid detection of changes in fluorescence as the circular DNAundergoes structural changes upon supercoiling and relaxation. Theinstant fluorophore-quencher (AT)_(n) nucleic acids can be used to gaugesuperhelicity of DNA molecules and detect the presence of DNAtopology-affecting enzymes. The instant fluorophore-quencher nucleicacids are well-suited for high-throughput analyses of topology changesof DNA because of the speed of change in DNA conformation and the fastkinetics of changes in fluorescence combined with the ease ofmass-production of the instant nucleic acid molecules.

Without wanting to be bound by theory, it is believed that the speed offluorescence change produced by the instant fluorophore-quencher nucleicacids can be explained by two mechanisms: fast extrusion of asupercoiling-dependent hairpin leading to effective quenching offluorescence due to optimized proximity of fluorophore and quencher inthe hairpin conformation combined with effective removal of quenchingdue to optimized distance between fluorophore and quencher in therelaxed double-stranded DNA molecule.

In preferred embodiments, the size of the (AT)_(n) sequence and thedistance between the fluorophore and quencher located on the same strandcombine fast interconversion between extruded and unextrudedconformation, which is promoted by shorter hairpin supporting (AT)_(n)sequences, with sufficient distance between fluorophore and quencher inthe double-stranded state to allow unquenching, which is supported bylonger (AT)_(n) sequences.

The instant fluorophore-quencher nucleic acids combine several featuresto provide an advantageous system for easy detection and screening forDNA topology-affecting enzymes: at least one fluorophore and at leastone quencher at a distance that is chosen to combine direct adjacency offluorophore and quencher in the stem of the extruded hairpin structurefor maximal quenching, sufficient distance in the relaxeddouble-stranded state for maximal unquenching, and ease ofmass-production due to the novel production method disclosed herein.

The circular double-stranded plasmids comprising the instantfluorophore-quencher nucleic acids can be made in small batches or inlarge milligram amounts. For both small and large batch preparation, adouble-stranded oligomer comprising an (AT)_(n) sequence and two nickingendonuclease recognition sites at each end is provided, wherein thenicking endonuclease recognition sites can be oriented in the samedirection, can be oriented in opposite directions, can be recognitionsites for the same nicking endonuclease, or can be recognition sites fordifferent nicking endonuclease; such oligomer is ligated to a pre-cutdouble-stranded DNA plasmid using standard cloning techniques, such thatthe nicking endonuclease recognition sites are preserved in thedouble-stranded DNA plasmid and flank the (AT)_(n) oligomer sequence.

It is contemplated that the (AT)_(n) sequence between the nickingendonuclease recognition sites of said plasmid undergoes structuralchanges including formation of hairpin structures and/or cruciformstructures upon supercoiling-dependent structural changes of thedouble-stranded DNA plasmid.

In specific embodiments, a fluorophore-quencher nucleic acid sequence isintroduced into the same strand of the (AT)_(n) sequence of thedouble-stranded circular plasmid by digesting the plasmid with a nickingenzyme that removes one DNA strand located between the two nickingendonuclease recognition sites. In some embodiments, a nucleic acidcontaining at least one fluorophore and at least one quencher conjugatedto dTs at a predetermined distance is ligated to the nicked sites of theplasmid using standard cloning techniques.

In other embodiments, a fluorophore-comprising nucleic acid sequence isintroduced into one strand and a quencher-comprising nucleic acidsequence is introduced into the opposite strand of the (AT)_(n) sequenceof the double-stranded circular plasmid, where the (AT)_(n) sequence ofthe double-stranded circular plasmid comprises two nicking sites for afirst nicking endonuclease on one strand and two nicking sites for asecond nicking endonuclease on the opposite strand. Following nicking ofthe first strand by a first nicking endonuclease, afluorophore-conjugated nucleic acid sequence can be ligated between thenicked ends. Subsequently, the opposite strand can be nicked by a secondnicking endonuclease and a quencher-conjugated nucleic acid sequence canbe ligated between the second nicked ends such that thefluorophore-comprising nucleic acid and the quencher-comprising nucleicacids are located on opposing strands.

For small batch preparation of the fluorophore-quencher comprisingcircular double-stranded DNA plasmids, the plasmids can be purified byagarose gel electrophoresis or Cesium chloride-ethidium bromideequilibrium gradient banding; however, yields using these methods arelimited because isolation of plasmid DNA from agarose gel pieces andextraction of DNA from Cesium chloride-ethidium bromide bands generallyresult in loss of DNA and, thus, these methods are not suitable for thepreparation of large quantities of circular plasmid DNA.

In one embodiment, the method disclosed herein combines the cloningstrategy described above employing the described nicking technique and asimple DNA recovery procedure, both of which enable a yield of, forexample, 50%, 60%, 70% or up to 80% or more, and, thus, enable thegeneration of milligram amounts of the fluorophore-quencher nucleic acidcomprising circular double-stranded plasmid DNA.

For the large scale production of relaxed fluorophore-quenchercomprising circular double-stranded DNA, milligram amounts of a circulardouble-stranded plasmid DNA comprising an (AT)_(n) sequence are digestedwith a nicking endonuclease and phosphorylated oligomer comprising theinstant fluorophore-quencher nucleic acid are added and incubated inappropriate conditions to allow annealing of the instantfluorophore-quencher nucleic acid to the single strand of plasmid DNAlocated between the nicked sites. DNA ligase is added to ligate thefluorophore-quencher nucleic acid to the nicked ends. It hassurprisingly been discovered that the yield of double stranded DNAcontaining the instant fluorophore-quencher nucleic acid can besignificantly increased when DNA polymerase and dNTPs are added to theligation reaction. It has also been determined that the yield can befurther increased when the reaction mixture after the ligation reactionis incubated with T5 exonuclease to digest single-stranded oligomer andnicked or gapped double-stranded starting material plasmid DNA.Advantageously, the T5 exonuclease does not digest double-stranded DNAsuccessfully ligated to the instant fluorophore-quencher nucleic acid.The yield of production of fluorophore-quencher comprising circulardouble-stranded DNA can further be increased by extracting the DNA withphenol twice, precipitating with isopropanol, washing with 70% ethanol,dissolving the DNA pellet and dialyzing the resultant DNA to obtainessentially pure and ready for use relaxed fluorophore-quenchercomprising circular double-stranded DNA.

For large scale production of supercoiled fluorophore-quenchercomprising circular double-stranded DNA, milligram amounts of a circulardouble-stranded plasmid DNA comprising an (AT)_(n) sequence are digestedwith a nicking endonuclease and phosphorylated oligomers comprising theinstant fluorophore-quencher nucleic acid are added and incubated inappropriate conditions to allow annealing of the instantfluorophore-quencher nucleic acid to the single strand of plasmid DNAlocated between the nicked sites. DNA ligase in the presence of ethidiumbromide is added to ligate the fluorophore-quencher nucleic acid to thenicked ends and the procedure as described above, including addition ofT5 exonuclease to remove single-stranded oligomers and nicked or gappeddouble-stranded starting material plasmid DNA, is performed.

Advantageously, the high yield of up to, for example, 80% obtained usingthe method of the subject invention allows the use of the instantplasmid DNAs to screen small-compound libraries containing millions ofcompounds using high throughput screening.

In specific embodiments, the double-stranded DNA plasmids comprising atleast one fluorophore and at least one quencher on the same stranddisplay maximal fluorescence in the relaxed state because the quencherand fluorophore located on the same strand are separated and noquenching occurs. In contrast, in the supercoiled state of the circulardouble-stranded plasmid, the fluorophore-quencher comprising (AT)_(n)nucleic acid strand interconverts from the unextruded to an extrudedconformation and due to the close proximity of fluorophore and quencherin the extruded conformation, fluorescence is quenched.

In other embodiments, the double-stranded DNA plasmids comprise at leastone fluorophore on one strand and at least one quencher on the oppositestrand and display no fluorescence in the relaxed double-stranded statebecause the fluorophore and quencher on opposite strands are in closeproximity such that the fluorescence of the fluorophore is quenched. Incontrast, in the supercoiled state of said circular double-strandedplasmids, when the fluorophore-comprising and the quencher-comprisingnucleic strands both interconvert from the unextruded to an extrudedconformation and form a cruciform structure, the fluorophore andquencher are in such distance as to prevent the quenching of fluorophorefluorescence.

It is contemplated that in some embodiments, the instant circulardouble-stranded plasmid comprising fluorophore-quencher nucleic acidscontain more than one fluorophore-quencher pair, whereas eachfluorophore-quencher pair is located in an (AT)_(n) nucleic acidsequence and forms a hairpin structure upon supercoiling of the circularDNA molecule. In other embodiments, the circular DNA molecule cancomprise more than one fluorophore and more than one quencher locatedwithin the same (AT)_(n) nucleic acid sequence, wherein the supercoilingof the circular DNA molecule induces a hairpin comprising more than onefluorophore and more than one quencher with each fluorophore being inclose proximity to a quencher in the hairpin conformation to quenchfluorophore fluorescence and each fluorophore and each quencher being ata sufficient distance in the relaxed double-stranded conformation toprevent quenching.

In specific embodiments, the (AT)_(n) sequence of the instantfluorophore-quencher nucleic acid can comprise a low of about 12 ATdinucleotides to a high of about 50 AT dinucleotides. For example, theinstant fluorophore-quencher nucleic acid can comprise AT dinucleotidesequences from about 12 ATs to about 17 ATs; about 18 ATs to about 25ATs; about 26 ATs to about 33 ATs; about 34 to about 41 ATs; or about 42to about 50 ATs.

In preferred embodiments, the fluorophore-quencher nucleic acidcomprises about 20 to about 25 AT dinucleotides. In a more preferredembodiment, the fluorophore-quencher nucleic acid comprises about 20 toabout 22 AT dinucleotides. In most preferred embodiments, thefluorophore-quencher nucleic acid comprises 21 AT dinucleotides.

The (AT)_(n) sequence of the instant nucleic acid can comprise the atleast one fluorophore and the at least one quencher conjugated to adeoxythymidine (dT) at a predetermined distance from the 5′ end of the(AT)_(n) sequence. For example, the fluorophore can be conjugated to adT located at a low distance of fourth position from the 5′ start of the(AT)_(n) sequence to a high distance of about fourteenth position fromthe 5′ start of the (AT)_(n) sequence. In specific embodiments, thefluorophore can be located at about the fourth; the fifth; the sixth;the seventh; the eighth; the ninth; the tenth; the eleventh; thetwelfth; the thirteenth; or the fourteenth position from the start ofthe (AT)_(n) sequence.

In some embodiments, the at least one quencher of thefluorophore-quencher nucleic acids can be conjugated to a dT located ata low distance of fourth position from the 5′ start of the (AT)_(n)sequence to a high distance of about fourteenth position. For example, aquencher can be localized at about the fourth; the fifth; the sixth; theseventh; the eighth; the ninth; the tenth; the eleventh; the twelfth;the thirteenth; or the fourteenth position from the start of the(AT)_(n) sequence of the fluorophore-quencher nucleic acid.

The fluorophore-quencher nucleic acid having a quencher at a location ofabout fourth to about fourteenth position from the 5′ start of the(AT)_(n) sequence can have a fluorophore located at a low distance ofabout 28th position from the 5′ start of the (AT)_(n) sequence to a highdistance of about the 40th position from the 5′ start. For example, afluorophore-quencher nucleic acid sequence can have a quencher at aboutthe fourth to the fourteenth position from the 5′ start of the (AT)_(n)sequence and a fluorophore at about the 28th; 29th; 30th; the 31st; the32nd; the 33rd; the 34th; the 35h; the 36th; the 37th; the 38th; the39th; or the 40th position from the 5′ start of the (AT)_(n) sequence.

The fluorophore-quencher nucleic acid having a fluorophore at a locationof about fourth to about fourteenth position from the 5′ start of the(AT)_(n) sequence can have a quencher located at a low distance of about28th position from the 5′ start of the (AT)_(n) sequence to a highdistance of about the 40th position. For example, a fluorophore-quenchernucleic acid sequence can have a quencher at about the fourth to thefourteenth position from the 5′ start of the (AT)_(n) sequence and afluorophore at about the 28th; 29th; 30th; the 31st; the 32nd; the 33rd;the 34th; the 35th; the 36th; the 37th; the 38th; the 39th; or the 40thposition from the 5′ start of the (AT)_(n) sequence.

It is contemplated that the location of the fluorophore andquencher-conjugated dT on the same strand within the (AT)_(n) sequenceis such that the proximity of fluorophore and quencher in an extrudedconformation provide maximal quenching and the fluorophore and quencherin an unextruded, double-stranded conformation provide maximalfluorescence of the fluorophore. For optimized detection of fluorescencechanges that allows utilization of the instant nucleic acids inhigh-throughput analyses, it is desirable that the fluorescence in thequenched conformation is as low as possible and the fluorescence in theunquenched conformation is as high as possible combined with the mostrapid interconversion from one conformation to the other.

In one embodiment, a distance between the fluorophore and quencherwithin the (AT)_(n) sequence of about 25 nucleotides in the instantfluorophore-quencher nucleic acid provides excellent FRET efficiency. Ithas also been determined that an increase in the distance betweenfluorophore and quencher to about 29 nucleotides leads to a significantreduction in FRET efficiency, whereas a reduction of the distance toabout 21 nucleotides leads to a less significant reduction.

In preferred embodiments, a fluorophore of the nucleic acid is locatedat about the 6th to about the 10th position from the 5′ start of an(AT)_(n) sequence comprising about 20 to 22 AT dinucleotides, (AT)₂₀₋₂₂,and a quencher is located at about the 32nd to the 36th position fromthe 5′ start of the (AT)₂₀₋₂₂ sequence. In a more preferred embodiment,a fluorophore of the nucleic acid is located at about the 8th to aboutthe 10th position from the 5′ start of an (AT)_(n) sequence comprisingabout 20 to 22 AT dinucleotides, (AT)₂₀₋₂₂, and a quencher is located atabout the 34th to the 36th position from the 5′ start of the (AT)₂₀₋₂₂sequence.

In a most preferred embodiment, a fluorophore is located at the 8thposition from the 5′ start of an AT sequence comprising 21 AT, (AT)₂₁,and a quencher is located at the 34th position from the 5′ start of the(AT)₂₁ sequence, whereby the fluorophore-conjugated dT and thequencher-conjugated dT are separated by 25 nucleotides.

Many fluorophores can be used to make the instant fluorophore-quenchernucleic acids. For example, the fluorophore can be 6-FAM (fluoroscein),Cy3™, TAMRA™, JOE, Cy5™, Cy5.5™, MAX, TET™, Carboxy-X-Rhodamine, TYE™563, TYE™ 665, TYE 705, Yakima Yellow®, Hexachlorofluorescein, TEX 615,Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 594,Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 750m 5′ IRDye® 700,5′IRDye® 800, 5′ IRDye®800CW, ATTO™ 488, ATTO™ 532, ATTO™ 550, ATTO™565, ATTO™ Rho101, ATTO™ 590, ATTO™ 633, ATTO™ 647, Rhodamine Green™-X,Rhodamine Red™-X, 5-TAMRA™, WEllRED D2, WellRED D3, WellRED D4, TexasRed®-X, Lightcycler® 640, DY 750, BODIPY FL, EDANS, or IAEDANS.

The quenchers used to make the instant fluorophore-quencher nucleicacids can be, for example, Dabcyl, DDQ-I, Eclipse, Iowa Black FQ, BHQ-1,QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, or BHQ-3.

Preferred fluorophore-quencher pairs useful in the instantfluorophore-quencher nucleic acids are shown in Tables 1 to 3 below.

TABLE 1 Fluorophore and quencher selection Max Max Fluorophoreabsorbance (nm) emission (nm) Quencher 6-FAM 494 521 Dabcyl/BHQ-1(Fluorescein) TET 521 536 Dabcyl/BHQ-1 HEX 535 556 Dabcyl/BHQ-1 Cy3 552570 BHQ-2 TAMRA 565 580 BHQ-2 Alexa Fluor 568 578 603 BHQ-2 Texas Red-X583 603 BHQ-2 Cy 5 646 667 BHQ-3 Alexa Fluor 680 679 702 BHQ-3 Cy5.5 683707 BHQ-3 Cy 7 743 767 BHQ-3

TABLE 2 Quencher spatial data Max Quenching Quencher absorbance (nm)range (nm) Dabcyl 453 380-530 BHQ-1 534 480-580 BHQ-2 579 560-650 BHQ-3650 620-730

TABLE 3 Common donor-acceptor pairs for FRET with Foster Radius (R0)Donor Acceptor R0 (Å) Fluorescein Tetramethylrhodamine 55 IAEDANSFluorescein 46 EDANS Dabcyl 33 Fluorescein Fluorescein 44 BODIPY FLBODIPY FL 57 Fluorescein QSY 7 61

The circular double-stranded DNA molecules comprising the instantfluorophore-quencher sequences can be used to detect and quantify thepresence of DNA topology affecting enzymes in a biological sample.

In a specific embodiment, the circular double-stranded DNA comprising atleast one fluorophore and at least one quencher on the same strandundergoes supercoiling in the presence of a DNA gyrase, wherein thefluorophore-quencher comprising nucleic acid sequence undergoes rapidlocalized DNA conformation transition, i.e. interconversion from theunextruded conformation in the double-stranded DNA to an extrudedconformation in the supercoiled state and quenching of fluorophorefluorescence occurs based on the close proximity of the fluorophore andquencher in the extruded conformation. In the presence of a DNAtopoisomerase, on the contrary, supercoiled circular DNA moleculescomprising the instant fluorophore-quencher nucleic acid on the samestrand undergo relaxation, wherein the fluorophore-quencher comprisingnucleic acid sequence undergoes localized DNA conformation transitionfrom the extruded conformation of the supercoiled state to theunextruded conformation of the relaxed, double-stranded state andfluorescence occurs because the fluorophore and quencher are located ina sufficient distance that prevents quenching.

Therefore, the instant fluorophore-quencher nucleic acid sequencepresent on the same stand of a circular DNA molecule can be used todetect DNA topology-affecting enzymes, including DNA topoisomerases, DNAgyrases, DNA nicking endonucleases, and DNA endonucleases, in a highlyefficient manner.

When the fluorophore-comprising nucleic acid sequence and thequencher-comprising nucleic acid sequence are present on oppositestrands of a circular DNA molecule, the presence of DNA gyrase can bedetected when relaxed circular plasmid DNA is supercoiled and thefluorophore and quencher positioned on opposite strands are located onthe ends or tips of cruciform structures formed in the extrudedconformation such that quenching cannot occur. The presence of DNAtopoisomerase, in contrast, can be detected when supercoiled circularplasmid DNA is relaxed and the fluorophore and quencher positioned onopposite strands are in close proximity such that the fluorescence ofthe fluorophore is quenched.

In specific embodiments, the fluorophore of the nucleic acid isfluorescein and the quencher is dabcyl. In more specific embodiments,the distance between fluorescein and the dabcyl-conjugated dT in the(AT)_(n) sequence can be as low as about 14 nucleotides to as high asabout 32 nucleotides. In preferred embodiments, the distance between thefluorescein and the dabcyl-conjugated dT is from about 14 to about 16nucleotides; from about 17 to about 20 nucleotides; from about 21 toabout 23 nucleotides; from 24 to about 26 nucleotides; and from about 27to about 32 nucleotides. In more preferred embodiments, the distancebetween fluorescein and dabcyl-conjugated dT is from about 21 to about25 nucleotides. In most preferred embodiments, the distance betweenfluorescein and dabcyl-conjugated dT is 25 nucleotides.

In one embodiment, the fluorescein fluorophore and the dabcyl quencherhaving a distance of about 88 Å to about 100 Å in the double-stranded,relaxed conformation of the instant nucleic acid, i.e., being conjugatedto dTs with about 25 nucleotides between the fluorescein-conjugated dTand the dabcyl-conjugated dT, results in high fluorescence offluorescein. In contrast, when the fluorescein fluorophore and thedabcyl quencher are in the extruded hairpin conformation the distancebetween fluorescein and dabcyl is about 20 Å and the fluorescence offluorescein is efficiently quenched by dabcyl. Advantageously, the FRETefficiency of the instant fluorescein-dabcyl comprising nucleic acidhaving about 25 nucleotides between the fluorescein-conjugated dT andthe dabcyl-conjugated dT is about 0.83.

In some embodiments, the fluorophore is TAMRA and the quencher is BHQ2and the distance between the TAMRA- and the BHQ2-conjugated dT in the(AT)_(n) sequence is 25 nucleotides.

It has been discovered that a fluorescein-dabcyl comprising nucleic acidhaving a reduced distance between fluorescein and dabcyl of about 21nucleotides can result in a FRET efficiency of about 0.75, whereas anincrease in the distance between fluorescein and dabcyl to about 29nucleotides can result in a FRET efficiency of only 0.51. It has furtherbeen discovered that the use of different fluorophore-quencher pairs,such as TAMRA and BHQ2, at the same optimized distance of 25 nucleotidesresults in a FRET efficiency of 0.8 similar to the FRET efficiency ofthe fluorescein-dabcyl comprising nucleic acid, albeit with a lowerfluorescence of the TAMRA fluorophore compared to fluorescein.

The FRET efficiency of the fluorophore and quencher of the instantnucleic acids can range from about 0.45 to about 0.90. For example, theFRET efficiency can be from about 0.45 to about 0.5; from about 0.51 toabout 0.59; from about 0.6 to about 0.69; from about 0.7 to about 0.79;from about 0.8 to about 0.82; from about 0.83 to about 0.85; from about0.86 to about 0.88 and from about 0.89 to about 0.9.

It has been discovered that a supercoiled fluorescein-dabcyl comprisingplasmid, pAB1_FL905, can be interconverted to a relaxed state byexposure to DNA topoisomerase with the fluorescein and dabcyl-dTs havingmaximal distance in the relaxed state and resulting in removal ofquenching with a half-maximal increase in fluorescence in about 25seconds.

It was further discovered that exposure of a supercoiledfluorescein-dabcyl-comprising plasmid, pAB1_FL905, to DNA nickingendonuclease can result in rapid nicking with a half-maximal increase influorescence of the nicked plasmid in about 15 seconds.

In further embodiments, the circular plasmid comprising afluorophore-quencher nucleic acid can have nicking endonucleaserecognition sites outside the fluorophore-quencher nucleic acidsequence, which second nicking endonuclease recognition sites differfrom the nicking endonuclease recognition sites of thefluorophore-quencher sequence. The second nicking endonucleaserecognition sites can be in the same orientation and can be recognitionsites for the same nicking endonuclease or for different endonucleases.In specific embodiments, the circular DNA plasmid comprising nickingendonuclease recognition sites outside the fluorophore-quenchercontaining nucleic acid sequence can be used to detect the presence ofnicking endonucleases in a sample, where in the presence of a nickingendonuclease the circular DNA plasmid is relaxed and a change influorescence occurs.

In other embodiments, the circular DNA plasmid comprising nickingendonuclease recognition sites outside the fluorophore-quencher nucleicacid sequence can be nicked with a nicking endonuclease and a RNAoligomer can be inserted between the nicked ends. The RNAoligomer-comprising plasmid can be used to detect RNAse activity in asample. For example, a supercoiled DNA plasmid comprising a RNA oligomercan be relaxed by exposure to an RNAse and the change in fluorescence inthe relaxed state plasmid is indicative of the presence of the RNAse.

In another embodiment, the fluorophore-quencher nucleic acid comprisingdouble-stranded plasmid can be used to detect DNA endonuclease activityin a sample. For example, the circular DNA plasmid comprising afluorophore-quencher containing nucleic acid sequence can be used in thesupercoiled state and exposed to a DNAse endonuclease, whichendonuclease cleaves the double-stranded DNA at specific endonucleaserecognition sites and the linearized plasmid DNA having the fluorophoreand quencher on the same strand and separated in the linearized statefluoresces, which fluorescence is indicative of the presence of the DNAendonuclease.

Advantageously, the instant circular DNA plasmids comprisingfluorophore-quencher containing nucleic acid sequences can be used todetect the presence of, and study the properties of, for example, DNAtopoisomerases, DNA gyrases, DNA nicking endonucleases, DNAendonucleases, and RNAses and the presence of inhibitors of DNAtopoisomerases, DNA gyrases, DNA nicking endonucleases, DNAendonucleases, and RNAses.

The instant circular DNA plasmids comprising fluorophore-quenchernucleic acid sequences can also be used to screen for inhibitors of DNAtopoisomerase, DNA endonuclease, and DNA nicking endonuclease activity.In one method, a sample suspected of containing an inhibitor of a DNAtopoisomerase, DNA endonuclease, or DNA nicking endonuclease is added toa DNA topoisomerase, DNA endonuclease, or DNA nicking endonuclease and asupercoiled DNA plasmid comprising a fluorophore-quencher nucleic acidsequence one the same strand and an increase in fluorescence in thesample is indicative of the presence of uninhibited DNA topoisomerase,DNA endonuclease, or DNA nicking endonuclease activity, whereas theabsence of fluorescence is indicative of the presence of an inhibitor ofthe DNA topoisomerase, DNA endonuclease, or DNA nicking endonuclease,respectively.

A suitable method for screening for inhibitors of RNAse activity in asample can use a supercoiled plasmid comprising a fluorophore-quenchernucleic acid sequence on the same strand and a RNA oligomer, exposingsaid plasmid to an RNAse and a sample suspected of containing aninhibitor of a RNAse and detect an increase in fluorescence in thesample when no inhibitor is present, i.e., the DNA is relaxed by theRNAse, and detect the absence of fluorescence in a sample where aninhibitor is present, i.e., the supercoiled DNA is not relaxed becausethe inhibitor inhibits the relaxing activity of the RNAse.

Also provided are kits for screening inhibitors of DNA topoisomerases,DNA gyrases, DNA endonucleases, DNA nicking endonucleases or RNAses. Thekit can comprise, for example, a circular double-stranded DNA plasmidcomprising the fluorophore-quencher nucleic acid on the same strand, aDNA topoisomerase, DNA endonuclease, DNA nicking endonuclease or RNAses,wherein the plasmid is in the supercoiled conformation and the kit isused to detect inhibitors of the DNA topoisomerase, DNA endonuclease,DNA nicking endonuclease or RNAse. The kit can further comprise acircular double-stranded DNA plasmid comprising the fluorophore-quenchernucleic acid, and a DNA gyrase, wherein the plasmid is in the relaxedconformation and the kit is used to detect inhibitors of the DNA gyrase.

The kits may further be used in the methods described herein. The kitsmay also include at least one reagent and/or instructions for their use.Also, the kits may include one or more containers filled with reagent(s)and/or one or more molecules of the invention. The kits may alsocomprise a control composition. In certain embodiments, the kits mayadditionally include reagents and means for detecting the labelsprovided on the molecules of the invention. The means of allowingdetection may be by conjugation of detectable labels or substrates, suchas fluorescent compounds, enzymes, radioisotopes, heavy atoms, reportergenes, luminescent compounds, etc. As it would be understood by thoseskilled in the art, additional detection or labeling methodologies maybe used in the kits provided. Further, any pair of fluorophores (donorfluorophore and acceptor fluorophore) that undergoes FRET can be used inthe kits.

Among the DNA topoisomerases that can be detected using the instantfluorophore-quencher nucleic acid comprising circular plasmid DNAs aretopoisomerases including type I; type IA; type IB; type HA; type IV;bacterial topoisomerases, including E. coli topoisomerase I, bacterialtopoisomerase IV, bacterial DNA gyrase; and human topoisomerases I andHa and other topoisomerase IA and IB topoisomerases, and othertopoisomerase IIA and IIB topoisomerases. The methods can also be usedto screen for yeast topoisomerase II, mammalian topoisomerase IIa andIIb, prokakryotic DNA topoisomerase III, yeast DNA topoisomerase III,mammalian DNA topoisomerase IIIa and IIIb, and poxvirus and vaccinia DNAtopoisomerases.

Among the RNAses that can be detected using the RNA oligomer-comprisingfluorophore-quencher comprising circular plasmid DNAs are RNAse H andother RNAses.

Among the DNA nicking endonucleases useful in the generation of theinstant fluorophore-quencher nucleic acid comprising circular plasmidsand/or which nicking endonucleases can be detected using the instantfluorophore-quencher nucleic acid comprising circular plasmid DNAs areNt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BtsI, Nb.BsrDI, Nt.AlwI, Nb.BbvCI,Nt.BbvCI, Nb.BsmI, Nt.BsmAI, Nb.Bpu10I and nonspecific nickingendonucleases, such as mung bean nuclease.

Among the DNA endonucleases that can be detected using the instantfluorophore-quencher nucleic acid comprising circular plasmid DNAs areany commercially available DNA endonucleases that cut double-strandedplasmid DNA and for which the double-stranded plasmid DNA contains anendonuclease recognition site.

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. It will be further understood that terms, such asthose defined in commonly used dictionaries, should be interpreted ashaving a meaning that is consistent with their meaning in the context ofthe relevant art and/or as otherwise defined herein.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting.

EXAMPLES Example 1—Materials and Methods

Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4DNA ligase were purchased from New England Biolabs (Beverly, Mass.,USA). E. coli DNA topoisomerase I was purified as describedpreviously¹⁹. The following synthetic oligonucleotides were purchasedfrom MWG-Biotech, Inc. (Huntsville, Ala.): FL882 (5′-CCCTCAGCCCGACAGCACGAGACGATATATATATATATATATATATATATATATATATATATATATGGGCCAACCAACCAGCCCCTCAGCG-3′) (SEQ ID NO:3), FL883(5′-GATCCGCTGAGGGGCTGGTTGGTTGGCCCATATATATATATATATATATATATATATATATATATATATATCGTCTCGTGCTGTCGGGCTGAGGGCATG-3′) (SEQ ID NO:4), FL905(5′-TCAGCCCGACAGCACGAGACGATATATA[Dab-dT]ATATATATATATATATATATATATA[FL-dT]ATATATATGGGCCAACCAACCAGCCCC-3′) (SEQ ID NO:5),FL919 (5′-TCAGCCCGACAGCACGAGACGATATA[Dab-dT]ATATATATATATATATATATATATATATA[Fl-dT]ATATATGGGCCAACCAACCAGCCCC-3′) (SEQ ID NO:6),FL920 (5′-TCAGCCCGACAGCACGAGACGATATATATA[Dab-dT]ATATATATATATATATATATA[Fl-dt]ATATATATATGGGCCAACCAACCAGCCCC-3′) (SEQ ID NO:7), andFL924 (5′-TCAGCCCGACAGCACGAGACGATATATA[BHQ2-dT]ATATATATATATATATATATATATA [TAM-dT]ATATATATGGGCCAACCAACCAGCCCC-3′) (SEQ IDNO:8) where Dab-dT, Fl-dT, BHQ2-dT, and TAM-dT represent dabcyl-dT,fluorescein-dT, BHQ2-dT, and TAMRA-dT, respectively. QIAquick NucleotideRemoval Kit and QIAquick Gel Extraction Kit were obtained from Qiagen,Inc (Valencia, Calif.).

Plasmids

Plasmid pAB1 (2,757 bp) was constructed by inserting a 95 bp syntheticDNA fragment FL_AT42 (the annealing product of FL882 and FL883) betweenthe SphI and BamHI sites of pUC18. DNA sequencing was used to verify theinserted DNA sequence.

Fluorescence Spectroscopy

Fluorescence measurements can be performed using an ISS, Inc., PC1 photocounting spectrofluorimeter with an excitation wavelength of 470 nm andbandwidth resolution of ±4 nm or a Biotek Synergy H1 Hybrid Plate Readerwith an excitation wavelength of 482 nm.

Molecular Modeling

DNA molecular models can be generated using HyperChem 8.0.

Example 2—Strategy to Construct Relaxed (Rx) and Supercoiled (SC)PAB1_FL905

Alternating adenine-thymine sequences [(AT)_(n)] undergo very rapidcruciform formation, as no detectable kinetic barrier prevents rapidinterconversion between extruded and unextruded conformations insupercoiled (sc) plasmid DNA templates (5). This property of [(AT)] wasutilized to monitor supercoiling changes of plasmid DNA templates.

A 82 nucleotide (nt) DNA oligomer, FL905, was designed that contains adabcyl (Dab)-labeled deoxythymidine (dT) at 29th position from the5′-end (the 8^(th) position of the AT sequence from the 5′end) and afluorescein (Fl)-labeled dT at 55th position from the 5′-end (the34^(th) position of the AT sequence from the 5′end (FIG. 1A and FIG. 2).FIG. 3A shows that FL905 has intrinsic fluorescence before EB staining.If the 42 nt AT sequence adopts the hairpin structure, both thefluorescein and dabcyl are in close proximity in the major groove (˜20Å; FIG. 1B). The fluorescence of fluorescein in this conformation isgreatly quenched. In contrast, when the 42 nt AT sequence adopts thedouble-stranded DNA form, the distance between the fluorescein anddabcyl is more than 88.4 Å for B-form DNA (26 bp×3.4 Å=88.4 Å) and canbe 100 Å (FIG. 1C). The fluorescence of fluorescein in thedouble-stranded DNA is not quenched. With increasing temperature, afour-fold fluorescence intensity change of fluorescein can be observedas the 42 nt AT hairpin structure is melted (FIG. 3B). Thus,fluorescence resonance energy transfer (FRET) can be used to study theinterconversion between extruded and unextruded conformations of FL905.

A circular plasmid, pAB1, can be constructed by inserting a syntheticdouble-stranded oligomer, FL_AT42 that carries the 42 bp AT sequence(FIG. 3C), between the SphI and BamHI sites of pUC18 (FIG. 3D). Thecircular plasmid pAB1 can also contain two nicking edonuclease Nt.BbvCIrecongnition sites in the same orientation. In this way, DNA oligomerFL905 can be inserted between the two Nt.BbvCI sites according togenerate the relaxed pAB1_FL905 (FIG. 4A). Supercoiled (sc) pAB1_FL905can be generated through the treatment of rx pAB1_FL905 with bacterialDNA gyrase in the presence of ATP (FIG. 4B). Rx and sc pAB1_FL905 arepowerful tools to study DNA topology and topoisomerases by FRET andinhibitors of DNA gyrases (Figures SA), inhibitors of DNA topoisomerases(FIG. 6), inhibitors of DNA nicking endonnucelases (FIG. 7), inhibitorsof RNAses (FIG. 9), inhibitors of DNA endonucleases (FIG. 10).

The circular plasmid pAB1 can also contain two nicking endonucleaseNt.BbvCI recongnition sites on one strand and two nicking endonucleaseNb.BbvCI recognition sites on the opposite strand. In this way, a DNAoligomer carrying a fluorophore can be inserted between the two Nt.BbvCIsites and a DNA oligomer carrying a quencher can be inserted between thetwo Nb.BbvCI sites (FIG. 11). Supercoiling of such plasmid results inhigh fluorescence and relaxation in low fluorescence (FIG. 12).

Example 3—Small and Large Scale Production of Relaxed and SupercoiledPlasmids (FIG. 13)

For the synthesis of relaxed (rx) and supercoiled (sc) pAB1_FL905,pAB1_FL919, pAB1_FL920, and pAB1_FL924 on a small scale of reaction, 10μg of pAB1 (˜5.7 μmol) can be digested by 25 units of Nt.BbvCI in 200 μLof 1× CutSmart Buffer (50 mM Potassium Acetate, 20 mM Tris-Acetate, 10mM Magnesium Acetate, 100 μg/mL BSA, pH 7.9). After the digestion, 80pmol of phosphorylated FL905 can be added into the reaction mixture; thereaction mixture can be incubated at 90° C. in a 4-liter water bath forone minute and then cooled down to room temperature in the water bath(˜4 to 5 hours or overnight). To generate rx pAB1_FL905, 300 units of T4DNA ligase can be added into the reaction mixtures in the presence of 10mM of DTT and 2 mM of ATP (final concentrations). The reaction mixturescan be incubated at 37° C. to seal the nicks and yield rx pAB1_FL905.The rx pAB1_FL905 can be separated by 1% agarose gel electrophoresis andpurified by QIAquick Gel Extraction Kit. Typically, ˜6 μg of rxpAB1_FL905 (˜60% yield) can be obtained. To produce sc pAB1_FL905, 1 μgof rx pAB1_FL905 can be treated with 5 units of E. coli DNA gyrase for 1hour at 37° C. The sc pAB1_FL905 can be purified by QIAquick NucleotideRemoval Kit or separated by 1% agarose gel and purified by QIAquick GelExtraction Kit. An alternative procedure can also be used to produce scpAB1_FL905. First, the annealed product of the Nt.BbvCI digested pAB1and FL905 can be purified by QIAquick Nucleotide Removal Kit. Thepurified DNA sample (˜1 μg) can be ligated with 300 units of T4 DNAligase in the presence of 5 units of DNA gyrase. The sc and rxpAB1_FL905 can be separated by using a 1% agarose gel and purified byusing QIAquick Gel Extraction Kit. Rx and sc pAB1_FL919, pAB1_FL920, andpAB1_FL924 can also be generated similarly.

For a typical large scale of reaction, 1 mg of pAB1 (˜570 pmol) can bedigested by 2500 units of Nt.BbvCI in 20 ml of 1× CutSmart Buffer forone hour at 37° C. After the digestion, 11,400 pmol of phosphorylatedFL905 can be added into the reaction mixture. The reaction mixture canbe incubated at 90° C. in a 4-liter water bath for two minutes and thencooled down to room temperature in the water bath (˜4 to 5 hours orovernight). To generate rx pAB1_FL905, 25,000 units of T4 DNA ligase(NEB) can be added into the reaction mixtures in the presence of 10 mMof DTT and 2 mM of ATP (final concentrations). The reaction mixtures canbe incubated at 37° C. to seal the nicks and yield rx pAB1_FL905. Toincrease the yield of rx pAB1_FL905, 750 units of T4 DNA polymerase and200 μM of dNTPs can be added to the reaction mixture. Then, 5,000 unitsof T5 exonuclease (NEB) can be added into the reaction mixture to digestsingle-stranded (ss) oligomer FL905 and nicked or gapped pAB1 except rxpAB1_FL905 (T5 exonuclease only digested ss FL905 and nicked pAB1 anddoes not degrade rx or sc pAB1_FL905). The rx pAB1_FL905 sample can beextracted with 20 mL of phenol twice, precipitated with 0.7 volume ofisopropanol, washed once with 70% ethanol, dissolved into 0.5 mL of 10mM Tris-HCl, pH 8.0, and dialyzed against 1,000 mL of 10 mM Tris-HCl, pH8.0. The rx pAB1_FL905 is essentially pure and ready for use. It wasobserved that on average ˜0.8 mg of rx pAB1_FL905 from 1 mg of pAB1 canbe generated (˜80% yield).

To generate sc pAB1_FL905, 25,000 units of T4 DNA ligase (NEB) can beadded into the reaction mixtures in the presence of 25 μM ethidiumbromide, 10 mM of DTT and 2 mM of ATP (final concentrations). Thereaction mixtures can be incubated at 37° C. to seal the nicks and yieldsc pAB1_FL905. To increase the yield of sc pAB1_FL905, 750 units of T4DNA polymerase and 200 μM of dNTPs can be added to the reaction mixture.Then, 5,000 units of T5 exonuclease (NEB) can be added into the reactionmixture to digest single-stranded (ss) oligomer FL905 and nicked orgapped pAB1 except sc pAB1_FL905. The sc pAB1_FL905 sample can beextracted with 20 mL of phenol twice, precipitated with 0.7 volume ofisopropanol, washed once with 70% ethanol, dissolved into 0.5 mL of 10mM Tris-HCl, pH 8.0, and dialyzed against 1,000 mL of 10 mM Tris-HCl, pH8.0. The sc pAB1_FL905 is essentially pure and ready for use. It wasobserved that on average 0.8 mg of sc pAB1_FL905 from 1 mg of pAB1 canbe generated (˜80% yield).

Usually it is required to purify rx and sc pAB1-FL905 by agarose gelelectrophoresis or CsCl-EB banding, which is very difficult to generatepure rx and sc pAB1_FL905 in the milligram range. The use of T5exonuclease followed by phenol extraction and isopropanol precipitationsignificantly simplifies the purification procedure. This proceduremakes the production of large amounts of rx and sc pAB1_FL905 feasible.Because of this procedure, it is possible to use rx or sc pAB1_FL905 tohigh throughput screen small compound libraries containing millions ofcompounds. Additionally, this new procedure also greatly increases theyield of rx and sc pAB1_FL905.

Example 4—Fluorescence Properties of Relaxed, Nicked, SupercoiledPAB1_FL905

The strategy shown in FIG. 13 can be used to produce large quantities ofrx pAB1-FL905. For example ˜0.5 mg of rx pAB1_FL905 and ˜0.6 mg of scpAB1_FL905 can be produced and purified by CsCl-EB equilibrium gradientbanding with approximately 60% yield. Rx and sc pAB1_FL905 haveintrinsic fluorescence before EB staining (FIG. 14D).

Fluorescence measurements can be performed using an ISS, Inc., PC1 photocounting spectrofluorimeter with an excitation wavelength of 470 nm andbandwidth resolution of ±4 nm or a Biotek Synergy H1 Hybrid Plate Readerwith an excitation wavelength of 482 nm.

When comparing fluorescence properties of sc, rx, and nicked (nk)pAB1_FL905, the fluorescence intensity of rx or nk pAB1_FL905 issignificantly higher than that of the sc pAB1_FL905 (FIG. 15A). Thekinetic results of pAB1_FL905 reacting with three different enzymes showthat Nt.BbvCI can quickly nick sc pAB1_FL905 with a half-life of 15seconds (FIG. 15B) continuing that (AT)_(n) can undergo very rapidhairpin and/or cruciform formation, as no detectable kinetic barrierprevents rapid interconversion between extruded and unextrudedconformations in sc plasmid DNA templates. This result demonstrates thatpAB1_FL905 is a good DNA substrate to study DNA topology andtopoisomerases by FRET.

Large amounts of E. coli DNA topoisomerase I can rapidly relax scpAB1_FL905 (FIG. 15C). The kinetics of E. coli DNA gyrase are relativelyslow (FIG. 15D).

Example 5—Fluorescence Properties of Relaxed, Nicked, SupercoiledPAB1_FL919 and PAB1_FL920

Similar fluorescence-labeled oligomers, FL919 and FL920, can begenerated that contain a dabcyl-labeled dT at 27th and 31^(st) position,and a fluorescein-labeled dT at 57th and 53th position from the 5′-end,respectively. In these oligomers, the distance between the dabcyl andfluorescein is different for FL905, FL919, and FL920. FL924 carries aBHQ2-labeled dT at 29th position from the 5′-end, i.e. the 8^(th)position of the AT sequence from the 5′end and a TAMRA-labeled dT at55th position from the 5′-end, i.e. the 34^(th) position of the ATsequence from the 5′end (FIGS. 17A-17B). These three oligomers can beinserted between the two Nt.BbvCI sites of pAB1 to yield rx and scpAB1_FL919, pAB1_FL920, and pAB1_FL924. Similar to pAB1_FL905, thefluorescence intensity of rx pAB1_FL919, pAB1_FL920, and pAB1_FL924 issignificantly higher than that of the sc DNA molecules (FIGS. 16A-16Dand FIGS. 17A-17B). However, the FRET efficiency of pAB1_FL919 andpAB1_FL920 is lower than that of pAB1_FL905 (FIG. 16A). Therefore, FL905has the optimal distance between dabcyl and fluorescein for studyingsupercoiling-dependent transitions of pAB1 by FRET. The fluorescenceintensity of pAB1_FL924 is lower than that of pAB1_FL905 although theFRET efficiency is similar for both DNA molecules.

Example 6—Gyrase Inhibition Assay

DNA gyrase inhibition assays can be performed in 50 μL of 1× gyrasebuffer (35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl₂, 2 mM DTT, 1.75 mM ATP, 5mM spermidine, 0.1 mg/mL BSA, 6.5% glycerol, pH7.5) containing 560 ng ofrx pAB1_FL905 and can be equilibrated to 37° C. 20 units of DNA gyrasecan be used to supercoil the rx pAB1_FL905 in the presence of differentconcentrations of novobiocin and ciprofloxacin (FIGS. 18A-18B). Thefluorescence intensity at λ_(em)=521 nm can be monitored with λ_(ex)=470nm in a microplate reader. The IC50 values can be estimated by nonlinearfitting of the following equation:

$F = {F_{\min} + \frac{F_{\max} - F_{\min}}{1 + 10^{{({{\log{({{IC}\; 50})}} - x})}P}}}$where F is the fluorescence intensity at the x concentration of aninhibitor. F_(max) and F_(min) are the maximum and minimum fluorescenceof the DNA sample, respectively. P is a slope parameter.

Example 7—Potential Applications

Rx or sc, fluorescently labeled pAB1_FL905 or similar DNA molecules canhave many potential applications. They can be used to studysupercoiling-dependent DNA topological changes or determine biochemicalproperties and kinetics of various DNA topoisomerases. In a preferredapplication, these DNA molecules can be used to screen inhibitors orcompounds targeting different DNA topoisomerases since many of thesecompounds are either anticancer drugs, such as doxorubicin, orantibiotics, such as ciprofloxacin. FIG. 5A shows a strategy foridentifying bacterial DNA gyrase inhibitors. In the absence of gyraseinhibitors, bacterial DNA gyrase is capable of converting rx DNAtemplates into sc DNA molecules (FIG. 5B). As demonstrated above, thisconversion results in quenching of fluorescence of pAB1_FL905. However,DNA gyrase inhibitors can inhibit this conversion. In this way, thefluorescence intensity of rx pAB1_FL905 is not changed. A titrationexperiment can yield an inhibition IC50 for the gyrase inhibitor.According to this strategy, titration experiments can be performed inwhich different concentrations of novobiocin and ciprofloxacin can beadded into DNA supercoiling assays. FIGS. 18A-18B shows the results fornovobiocin and ciprofloxacin, both of which can potently inhibit theactivities of DNA gyrase with an estimated IC50 of 0.48±0.14 and2.57±1.62 μM, respectively. Agarose gel electrophoresis can be used toconfirm that these antibiotics indeed potently inhibit DNA gyrasesactivities FIG. 5B. Due to simplicity, this FRET assay can be easilyadapted to a high throughput format to identify gyrase inhibitors frommillions of compounds found in small molecule libraries. Similar assayscan be used to identify inhibitors targeting other DNA topoisomerase,such human DNA topoisomerase I and II.

The instant methods can be used, for example, in antibiotic screeningkits to screen for inhibitors that target bacterial DNA gyrase. TheseDNA gyrase inhibitors can be developed into potent antibiotics. Thescreening kits can include, for example, the following components:relaxed pAB1_FL905 or a similar product, E. coli DNA gyrase, 5×DNAgyrase buffer, ATP, and novobiocin and/or ciprofloxacin as positivecontrol.

Antibiotic screening kits can also be used to screen for inhibitors thattarget bacterial DNA topoisomerase I. The screening kits can include,for example, the following components: supercoiled pAB1_FL905 or asimilar product, E. coli DNA topoisomerase I, and 10×DNA topoisomerase Ibuffer. Screening kits can also be used to screen for anticancer drugsthat target human DNA topoisomerase I. The screening kits can include,for example, the following components: supercoiled pAB1_FL905 or asimilar product, human DNA topoisomerase I, and 10×DNA topoisomerase Ibuffer. Finally, screening kits can be used to screen for anticancerdrugs that target human DNA topoisomerase II. The screening kits caninclude, for example, the following components: supercoiled pAB1_FL905or a similar product, human DNA topoisomerase II, and 10×DNAtopoisomerase I buffer.

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes in light thereof willbe suggested to persons skilled in the art and are included within thespirit and purview of this application. In addition, any elements orlimitations of any invention or embodiment thereof disclosed herein canbe combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

REFERENCES

-   1. Roychoudhury, S., Makin, K. M., Twinem, T. L., Stanton, D. T.,    Nelson, S. L., & Catrenich, C. E. (2003) Development and use of a    high-throughput bacterial DNA gyrase assay to identify mammalian    topoisomerase II inhibitors with whole-cell anticancer activity. J.    Biomol. Screen. 8, 157-163.-   2. Maxwell, A., Burton, N. P., & O'Hagan, N. (2006) High-throughput    assays for DNA gyrase and other topoisomerases. Nucleic Acids Res.    34, e104.-   3. Shapiro, A., Jahic, H., Prasad, S., Ehmann, D., Thresher, J.,    Gao, N., & Hajec, L. (2010) A homogeneous, high-throughput    fluorescence anisotropy-based DNA supercoiling assay. J. Biomol.    Screen. 15, 1088-1098.-   4. Jude, K. M., Hartland, A., & Berger, J. M. (2013) Real-time    detection of DNA topological changes with a fluorescently labeled    cruciform. Nucleic Acids Res. 41, e133.-   5. Greaves, D. R., Patient, R. K., & Lilley, D. M. (1985) Facile    cruciform formation by an (AT) 34 sequence from a Xenopus globin    gene. J Mol. Biol. 185, 461-478.

The invention claimed is:
 1. A method of screening for inhibitorstargeting DNA topoisomerases, DNA gyrases, DNA endonucleases, or DNAnicking endonucleases, the method comprising: providing a samplesuspected of containing an inhibitor of a DNA topoisomerase, DNA gyrase,DNA endonuclease, or DNA nicking endonuclease; adding a circularsupercoiled double-stranded plasmid comprising a nucleic acid sequencecomprising an adenosine-thymidine dinucleotide repeat (AT)_(n) sequence,characterized in that n is at least 12, the (AT)_(n) sequence comprisesat least one fluorophore and at least one quencher, each conjugated to adeoxythymidine (dT), and the fluorophore-conjugated dT and thequencher-conjugated dT are separated by at least 14 nucleotides and arelocated on the same DNA strand to the sample suspected of containing aninhibitor of a DNA topoisomerase, DNA endonuclease, or DNA nickingendonuclease; or adding a circular relaxed double-stranded plasmidcomprising a nucleic acid sequence comprising an adenosine-thymidinedinucleotide repeat (AT)_(n) sequence, characterized in that n is atleast 12, the (AT)_(n) sequence comprises at least one fluorophore andat least one quencher, each conjugated to a deoxythymidine (dT), and thefluorophore-conjugated dT and the quencher-conjugated dT are separatedby at least 14 nucleotides and are located on the same DNA strand to thesample suspected of containing an inhibitor of a DNA gyrase, adding aDNA topoisomerase, DNA endonuclease, or DNA nicking endonuclease to thesample containing the circular supercoiled double-stranded plasmid; oradding a DNA gyrase to the sample containing the circular relaxeddouble-stranded plasmid; quantifying fluorescence in the sample; andquantifying the amount of inhibitor present in the sample based on thefluorescence measured in the sample compared to the fluorescencemeasured in a control sample containing only the DNA topoisomerase, DNAendonuclease, or DNA nicking endonuclease and the circular supercoileddouble-stranded plasmid or containing only the DNA gyrase and thecircular relaxed double-stranded plasmid; characterized in that thefluorescence in the control sample containing the DNA topoisomerase, DNAendonuclease, or DNA nicking endonuclease is high due to theinterconversion of the circular supercoiled double-stranded plasmid to arelaxed conformation in the presence of the DNA topoisomerase, DNAendonuclease, or DNA nicking endonuclease; and the fluorescence in thesample is lower than in the control sample if the sample contains aninhibitor of the DNA topoisomerase, DNA endonuclease, or DNA nickingendonuclease; or characterized in that the fluorescence in the controlsample containing the DNA gyrase is low due to the interconversion ofthe circular relaxed double-stranded plasmid to a supercoiledconformation in the presence of the DNA gyrase; and the fluorescence inthe sample is higher than in the control sample if the sample containsan inhibitor of the DNA gyrase.
 2. The method, according to claim 1,comprising: adding a circular supercoiled double-stranded plasmid or acircular relaxed double-stranded plasmid both comprising a nucleic acidsequence comprising an (AT)_(n) sequence, characterized in that n is atleast 12, the (AT)_(n) sequence comprises at least one fluorophore andat least one quencher, each conjugated to a deoxythymidine (dT), and thefluorophore-conjugated dT and the quencher-conjugated dT are separatedby at least 14 nucleotides and are located on the opposite DNA strand.3. The method according to claim 1, comprising: adding a circularsupercoiled double-stranded plasmid or a circular relaxeddouble-stranded plasmid both comprising a nucleic acid sequencecomprising an (AT)_(n) sequence, characterized in that n is at least 12,the (AT)_(n) sequence comprises at least one fluorophore and at leastone quencher, each conjugated to a deoxythymidine (dT), and thefluorophore-conjugated dT and the quencher-conjugated dT are separatedby 25 nucleotides and are located on the same DNA strand.
 4. The method,according to claim 1, comprising: adding a circular supercoileddouble-stranded plasmid or a circular relaxed double-stranded plasmidboth comprising a nucleic acid sequence comprising an (AT)_(n) sequence,characterized in that n is at least 12, the (AT)_(n) sequence comprisesat least one fluorophore and at least one quencher, each conjugated to adeoxythymidine (dT), and the fluorophore-conjugated dT and thequencher-conjugated dT are separated by 25 nucleotides and are locatedon the opposite DNA strand.
 5. The method according to claim 1,characterized in that a half-maximal increase in fluorescence in thecontrol sample containing only a DNA topoisomerase occurs within 30seconds.
 6. The method according to claim 1, characterized in that ahalf-maximal increase in fluorescence in the control sample containingonly a DNA nicking endonuclease occurs within 20 seconds.
 7. A method ofscreening for inhibitors targeting RNAses, the method comprising:providing a sample suspected of containing an inhibitor of a RNAse;adding a circular supercoiled double-stranded plasmid comprising anucleic acid sequence comprising an adenosine-thymidine dinucleotiderepeat (AT)_(n) sequence, characterized in that n is at least 12, the(AT)_(n) sequence comprises at least one fluorophore and at least onequencher, each conjugated to a deoxythymidine (dT), and thefluorophore-conjugated dT and the quencher-conjugated dT are separatedby at least 14 nucleotides and are located on the same DNA strand, andfurther comprising a nucleic acid sequence comprising at least one RNAoligomer located on the same strand as the at least one fluorophore andthe at least one quencher; adding a RNAse; quantifying fluorescence inthe sample; and quantifying the amount of inhibitor present in thesample based on the fluorescence measured in the sample compared to thefluorescence measured in a control sample containing only the RNAse andthe circular supercoiled double-stranded plasmid; characterized in thatthe fluorescence in the control sample is high due to theinterconversion of the circular supercoiled double-stranded plasmid to arelaxed conformation in the presence of the RNAse; and the fluorescencein the sample is lower than in the control sample if the sample containsan inhibitor of the RNAse.
 8. The method, according to claim 7,comprising: adding a circular supercoiled double-stranded plasmidcomprising a nucleic acid sequence comprising an (AT)_(n) sequence,characterized in that n is at least 12, the (AT)_(n) sequence comprisesat least one fluorophore and at least one quencher, each conjugated to adeoxythymidine (dT), and the fluorophore-conjugated dT and thequencher-conjugated dT are separated by at least 14 nucleotides and arelocated on the opposite DNA strand.
 9. The method according to claim 7,comprising: adding a circular supercoiled double-stranded plasmidcomprising a nucleic acid sequence comprising an (AT)_(n) sequence,characterized in that n is at least 12, the (AT)_(n) sequence comprisesat least one fluorophore and at least one quencher, each conjugated to adeoxythymidine (dT), and the fluorophore-conjugated dT and thequencher-conjugated dT are separated by 25 nucleotides and are locatedon the same DNA strand.
 10. The method, according to claim 7,comprising: adding a circular supercoiled double-stranded plasmidcomprising a nucleic acid sequence comprising an (AT)_(n) sequence,characterized in that n is at least 12, the (AT)_(n) sequence comprisesat least one fluorophore and at least one quencher, each conjugated to adeoxythymidine (dT), and the fluorophore-conjugated dT and thequencher-conjugated dT are separated by 25 nucleotides and are locatedon the opposite DNA strand.
 11. The method, according to claim 7,comprising: adding a circular supercoiled double-stranded plasmidcomprising a nucleic acid sequence comprising at least one RNA oligomerlocated on the opposite strand as the at least one fluorophore and theat least one quencher.